We have developed a novel approach to identifying cis-acting sequences and trans-acting factors that are required to activate DNA replication and gene expression at the beginning of mammalian development. Plasmid DNA encoding a sensitive reporter gene linked to eukaryotic regulatory sequences is injected into the nuclei of mouse oocytes, fertilized eggs (1-cell embryos) and 2-cell embryos (where zygotic gene activation occurs). The results of such studies have revealed that replication and expression of genes encoded in the injected, extrachromosomal DNA responded to the same signals that regulate these functions in cellular DNA: They required specific cis-acting regulatory sequences and the trans-acting proteins that activate them, and they occurred only when the host cell executed the same function on its own genome. These results, taken together with analyses of endogenous gene expression and results from nuclear transplantation studies, revealed several novel features of zygotic gene expression at the beginning of mammalian development. A time dependent mechanism ("zygotic clock") delays transcription of zygotic genes and translation of nascent mRNA until the 2-cell stage in development. This provides an opportunity to remodel chromatin structure of parental genomes and possibly facilitate genomic imprinting without accidentally exposing genes to premature expression. In addition, the ability to use enhancers to activate promoters or replication origins does not appear until formation of 2-cell embryo, because an enhancer specific co-activator is absent prior to this stage in development. Therefore, the first two S-phases in mouse development occur under conditions where enhancers cannot function, a fact that may contribute to developmental acquisition of site specific initiation of DNA replication. The need for a TATA-box to facilitate promoter activity appears only after cell differentiation occurs and then only as a mediator of enhancer function. Prior to activation of zygotic genes in 2-cell embryos, the activity of promoters and replication origins is repressed by changes in chromatin structure, allowing positive regulatory factors to activate genes selectively. One of the first transcription factors to be expressed at the beginning of mouse development is TEAD2, one member of a new four gene family expressed throughout mammalian development. TEAD2 is expressed specifically during formation of a 2-cell embryo and is able to stimulate strongly the activity of appropriate enhancers, presumably turning on the next cascade of genes in the developmental program.